Tissue Collection Video Protocol

By now you should have taken pictures of the mushroom in the field, collected the mushroom, and brought it back for preservation. Your voucher data card should be completely filled out and your sample should have a collection number. We will now be taking a small piece of tissue from the mushroom and preserving it in our 1.5 mL eppi tube.

Lay your mushroom sample out on a clean table. Sterilize tweezers with an alcohol swab and lighter. (The primary goal with the flame is to burn off the alcohol, rather than getting the tweezers red-hot between each sample.) A quick wipe with a swab and a very brief flame (2-3 seconds) is sufficient.

We will begin by creating a "split" of the sample. Along with your tubes, you were sent small plastic bags for each sample. You will be placing a small portion of your collection in this bag. First, label the bag with your mycoflora number (or other collection number). If there are multiple specimens in the collection, place one of the smaller specimens in this bag. If there is only a single mushroom, place a small portion of the specimen (containing the sterile surface - gills or pores) into the bag. The piece does not need to be larger than a quarter. For very small samples, where the total collection would be signficantly reduced or damaged by creating a split, you may skip the process of creating a split of the collection, and retain the entire collection for shipment to your herbarium.

Tweeze off a small portion of the mushroom tissue from your split that is about the size of a grain of rice. Place this piece of material into your eppi tube and close the cover. Ensure that the tube is properly labeled with the collection number. Utilizing the small number in the lower right of your field data slip, trim the number with scissors to remove excess border, and tape it towards the top of the label with clear scotch tape. Try to leave the bottom of the tube uncovered so the sample is visible. That is all that needs to be done for this sample. Now you can "sterilize" your scalpel and tweezers (as described above) to repeat this process for your next sample.

It is possible to take dried tissue from any part of the mushroom in order to acquire DNA. For larger specimens, we typically suggest samples to be taken from the gills. For small samples, we recommend taking tissue from the stem of the mushroom as to preserve as much of the cap as possible. The cap contains many more parts that could be important for future microscopic study. Thus, we want to maintain as much of it as possible intact.

(One additional note: Projects that are utilizing Duke University in North Carolina as their primary herbarium can send the specimens in-tact, without creating the split.)

Saving Tissue Video Protocol - FTA Cards

This video protocol outlines a secondary method of tissue collection that works with fleshy fungi. This protocol is significantly more expensive than our preffered method, but it may be best in some individual circumstances. Please contact us if you would be interested in processing your tissue using this method.

Saving the Tissue – FTA Cards

Another common method for DNA preservation of wild mushroom samples are Whatman FTA Plant Cards. These are cards made of a paper that contains DNA stabilizing agents. The process to use the cards is simple.

Place a small sample of mushroom tissue in a circle on the interior of the card. Once again, you want to get a section of tissue that is from the interior flesh of the mushroom. This will prevent contamination during the DNA extraction process.

Fold the cover of the card over the tissue.

Hit the area containing the tissue with a hammer to macerate the tissue and release the interior liquid into the paper of the card.

Turn the card over to ensure there is a spot of moisture from the tissue that has soaked into the paper.

Remove any excess issue from inside the fold or that may be remaining on the cover. Try not to scratch or damage card material during this process.

Label your card with the collection number for this species.

Allow the card to dry.

While this is not recommended as our primary procedure, it will work very well for both getting a sample of DNA from the tissue and preserving it for future analysis. The cards are somewhat expensive at about $4 or $5 each. This will run you about $1.00 per sample for DNA collection. It is possible to put two or more samples into each section of the card, lowering the overall cost per sample. One advantage to these cards is their compact size, allowing for easier transportation. The primary disadvantage is that it is only possible to take samples from fleshy fungi. Whatman cards do not work well for dry or tough specimens (like polypores).

About North American Mycoflora Project

NAMP is a 501(c)(3) non-profit organization. We are working towards a single goal - the development of the first comprehensive mycoflora of North America. This project is a consortium of citizen scientists and professional mycologists performing a biological survey of all the macrofungi that occur in North America.